Tumor necrosis factor-α synthesis inhibitor, 3,6'-dithiothalidomide, reverses behavioral impairments induced by minimal traumatic brain injury in mice

Abstract

Mild traumatic brain injury (mTBI) patients do not show clear structural brain defects and, in general, do not require hospitalization, but frequently suffer from long-lasting cognitive, behavioral and emotional difficulties. Although there is no current effective treatment or cure for mTBI, tumor necrosis factor-alpha (TNF-α), a cytokine fundamental in the systemic inflammatory process, represents a potential drug target. TNF-α levels increase after mTBI and may induce or exacerbate secondary damage to brain tissue. The present study evaluated the efficacy of the experimental TNF-α synthesis inhibitor, 3,6'-dithiothalidomide, on recovery of mice from mTBI in a closed head weight-drop model that induces an acute elevation in brain TNF-α and an impairment in cognitive performance, as assessed by the Y-maze, by novel object recognition and by passive avoidance paradigms at 72 h and 7 days after injury. These impairments were fully ameliorated in mice that received a one time administration of 3,6'-dithiothalidomide at either a low (28 mg/kg) or high (56 mg/kg) dose provided either 1 h prior to injury, or at 1 or 12 h post-injury. Together, these results implicate TNF-α as a drug target for mTBI and suggests that 3,6'-dithiothalidomide may act as a neuroprotective drug to minimize impairment.

Figure 1. 3,6’-dithiothalidomide lowers TNF-α protein and nitrite generation in LPS-challenged RAW 264.7 cells without loss of viability

The effects of 3,6’-dithiothalidomide (3,6’-DT) and equimolar thalidomide (TH) on cell viability and LPS-induced elevation of TNF-α protein levels and nitrite generation in RAW 264.7 cells. Cells were incubated for 48 hr after administration of LPS with and without drug. Whereas 3,6’-dithiothalidomide (10 and 30 μM) significantly lowered TNF-α protein and nitrite generation, thalidomide (10 μM) significantly elevated generation of nitrite. Control refers to 100% DMSO.(n=4 observations. *, *** refers to p 0.05 and p 0.001 compared to control, respectively (Dunnett’s t test)).

Figure 2. 3,6’-dithiothalidomide lowers LPS-induced TNF-α levels in plasma and CNS

(A) Systemically administered LPS induced a rapid, time-dependent 3 log elevation in plasma TNF-α levels that peaked at 90 min. (B) This LPS-induction in plasma TNF-α levels was substantially (up to 80%) inhibited by prior administration of 3,6’-dithiothalidomide (3,6’-DT, 56 mg/kg) – levels are expressed as a percent of those determined at the same time in LPS alone animals. (C) Systemically administered LPS elevated CNS levels of TNF-α by 20% compared to basal levels in pure controls (no LPS). This CNS LPS-induced TNF-α rise was fully blocked by prior adminsitration of 3,6’-dithiothalidomide (56 mg/kg). (n=6. **, *** refers to p 0.01 and p 0.001 compared to LPS alone determined at the same time points, respectively (paired Student’s t test)).

Figure 3. mTBI induces impairment in performance in a Y maze preference index paradigm that is fully ameliorated by 3’6-dithiothalidomide

Performace of mice was quantitatively assessed in a Y maze paradigm at 72 hr and 7 days following mTBI as a preference index, calculated as (time new-time old)/(time new+time old). Values are mean ± SEM. (Upper) 72 hr post injury the mTBI group displayed a trend toward lower memory ability that did not reach significance (one-way ANOVA showed no differences; N.S) but this trend was not evident in mTBI drug-treated mice. (Lower) 7 days post injury, one way ANOVA showed that mTBI animals had a deficit in spatial memory performance compared with all the other groups (*p 0.05). No differences were found between any of the other groups, suggesting complete amelioration by 3’6-dithiothalidomide.

Figure 4. mTBI induces impairment in performance in a novel object recognition preference index paradigm that is fully ameliorated by 3’6-dithiothalidomide

Performace of mice was quantitatively measured in a novel object recognition paradigm at 72 hr and 7 days post mTBI as a preference index, calculated as (time new-time old)/(time new+time old). Values are mean ± SEM. (Upper) 72 hr post injury the mTBI group displayed a trend toward a lower preference index that did not reach significance (one-way ANOVA showed no differences; N.S) but this trend was not evident in mTBI drug-treated mice. (Lower) 7 days post injury, one way ANOVA showed that mTBI animals demonstrated a deficit in spatial memory performance compared with all the other groups (*p 0.05). No differences were found between any of the other groups, suggesting a complete amelioration by 3’6-dithiothalidomide.

Figure 5. mTBI induces a trend towards impairment in learning as assessed by passive avoidance that is not evident in mice administered 3’6-dithiothalidomide

Performace of mice was measured in a passive avoidance paradigm at 72 hr and 7 days post mTBI with learning defined as the percent of animals that did not enter the dark compartment on the second day. Values are in percents. (Upper). 72 hours post injury Chi square tests revealed no differences between all groups. (Lower). 7 days post injury Chi square tests revealed no differences between all groups, although a trend towards decreased learning was evident in mTBI mice that was not seen in animals treated with 3,6’dithiothalidomide prior to or post injury.